analysis of protein complexes by mass spectrometry
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Analysis of Protein Complexes by Mass
SpectrometryJohn Yates
The Scripps Research InstituteLaJolla, CA
Protein Complex Discovery
• Who: identity of proteins in complex?
• What: biological process involved?• Where: is the complex localized?• When: are proteins involved in the
complex?• How much: stoichiometry of proteins in
complex, quantity- relative vs absolute• Regulation: modifications (kinase,etc)
proteolysis (protease)
General Strategy for Protein CharacterizationGeneral Strategy for Protein Characterization
Purification/Purification/EnrichmentEnrichment
1-DE1-DE 2-DE2-DE SolutionSolution
• IdentificationIdentification• SequencingSequencing
PeptidesPeptidesProteinProtein oror
MeasurementMeasurement
AnalysisAnalysis
Mass SpectrometryMass Spectrometry
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2000020000
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Co
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800 800 1000 1000 1200 1200 1400 1400 1600 1600 1800 1800 2000 2000 Mass (m/z)Mass (m/z)
1406.72201406.7220
……PPGTGKTLLAK PPGTGKTLLAK AVANESGANFISVKAVANESGANFISVK FYVINGPEIM... FYVINGPEIM...
Molecular WeightMolecular Weight
200200 300300 400400 500500 600600 700700 800800 900900 10001000 12001200 14001400m/zm/z
00101020203030404050506060707080809090
100100
Rel
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bund
ance
Rel
ativ
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ance
922.4922.4
835.4835.4
1051.61051.6778.5778.5333.1333.1 619.0619.0 1074.51074.5 1236.71236.7468.1468.1 961.4961.4
FragmentationFragmentation
Post Translational RegulationPost Translational Regulation
• What structural changes occur to create What structural changes occur to create an active protein, alternate splicing, an active protein, alternate splicing, proteolytic processing?proteolytic processing?
• How is a protein’s activity regulated?How is a protein’s activity regulated?• Are modifications involved in regulation? Are modifications involved in regulation?
Protein Separation methods for ProteomicsProtein Separation methods for Proteomics
Dynamic range is central issue for Dynamic range is central issue for separationsseparations
• Gel ElectrophoresisGel Electrophoresis– 1 and 2-Dimensional Separations1 and 2-Dimensional Separations– Native and DenaturingNative and Denaturing– Detection- stainsDetection- stains
• Chromatographic or Electrophoretic Chromatographic or Electrophoretic – Liquid ChromatographyLiquid Chromatography– Capillary ElectrophoresisCapillary Electrophoresis– Affinity ChromatographyAffinity Chromatography– Multi-Dimensional SeparationsMulti-Dimensional Separations– DetectionDetection
Mass Spectrometry Analysis of ProteinsMass Spectrometry Analysis of Proteins
• Analysis of Peptides – digested proteins & mixtures of proteins Analysis of Peptides – digested proteins & mixtures of proteins (“Bottom Up” Approach)(“Bottom Up” Approach)– ESI – Tandem Mass Spectrometers (QIT, LIT, Q-TOFs, TSQs)ESI – Tandem Mass Spectrometers (QIT, LIT, Q-TOFs, TSQs)– MALDI-Tandem Mass Spectrometers (LIT, QIT, Q-TOFs, TOF/TOFs)MALDI-Tandem Mass Spectrometers (LIT, QIT, Q-TOFs, TOF/TOFs)– ESI-FTMS ESI-FTMS – MALDI-FTMSMALDI-FTMS
• Analysis of Intact Proteins (“Top Down” Approach)Analysis of Intact Proteins (“Top Down” Approach)– FTMSFTMS– ESI-TOFESI-TOF– MALDI-TOFMALDI-TOF
• Analyis of Protein ComplexesAnalyis of Protein Complexes– Ion Mobility mass spectrometersIon Mobility mass spectrometers– GEMMAGEMMA
• Mass Spectrometry technology evolves at a constant rateMass Spectrometry technology evolves at a constant rate– Product cycles are 18-24 monthsProduct cycles are 18-24 months
Computational ProteomicsComputational Proteomics
o Mass Spectrometry (MS/MS) data pre-processingMass Spectrometry (MS/MS) data pre-processingSequence SignaturesSequence SignaturesModification SignaturesModification SignaturesSpectral qualitySpectral quality
o Protein and Modification IdentificationProtein and Modification IdentificationWell established methods existsWell established methods exists
• Faster, More Sensitive, More AccurateFaster, More Sensitive, More AccurateHigh throughput and large scale High throughput and large scale de novode novo analysis analysis
o Global Quantification SoftwareGlobal Quantification SoftwareSoftware is needed based on established rules for isotopomer Software is needed based on established rules for isotopomer analysisanalysis
o Statistical Significance of MatchesStatistical Significance of Matcheso Empirical v. Non-empirical methodsEmpirical v. Non-empirical methods
Analytical Challenges
• Cell biology techniques to isolate structures• Sensitivity• Dynamic range: low affinity binders• Throughput
– Biochemical Throughput– Analytical Throughput
• Direct measurement of intact complex• Quantitation of components and
modifications
Comprehensive Analysis of Protein-Protein InteractionsComprehensive Analysis of Protein-Protein Interactions
Co-immunoprecipitation Co-immunoprecipitation
ProteolysisProteolysisLC/MS/MSLC/MS/MS
LC/LC/MS/MSLC/LC/MS/MS
Identification of Protein ComponentsIdentification of Protein ComponentsIdentification of ModificationsIdentification of Modifications
Dynamics of components and modificationsDynamics of components and modifications
Multiprotein ComplexMultiprotein ComplexProtein Interaction Protein Interaction ChromatographyChromatography
AgaroseAgarose ProteinGST
AgaroseAgarose Ig-G
AgaroseAgarose Ig-G
C
LTEV
TAP-Tagged Proteins
Cell Biology/Genetics
Second Generation Proteomics TechnologySecond Generation Proteomics TechnologyShotgun ProteomicsShotgun Proteomics
Identification of Proteins in MixturesIdentification of Proteins in Mixtures
MS/MSMS/MS
SEQUESTSEQUEST
ymr130ymr130 ymr142ymr142ymr154ymr154 ymr201ymr201
DigestionDigestion SeparationSeparation
Complex PeptideComplex PeptideMixtureMixture
LCLC
Protein Identification data acquired at ~1 peptide per 1-3 secsProtein Identification data acquired at ~1 peptide per 1-3 secs
Eng, McCormack, Yates, JASMS 1994Eng, McCormack, Yates, JASMS 1994
Integrated Multi-Integrated Multi-Dimensional Liquid Dimensional Liquid ChromatographyChromatography
100 micron ID100 micron ID 5 micron Tip
200 nL/min
SolventFlow
MS-MS of Peptide MixturesMS-MS of Peptide Mixtures
LCLC
MSMS
MS/MSMS/MS
1410.61410.6
DatabaseDatabase
IIGHFYDDWCPLKIIGHFYDDWCPLK
SPAFDSIMAETLKSPAFDSIMAETLKAFDSLPDDIHEKAFDSLPDDIHEK
GGILAQSPFLIIKGGILAQSPFLIIK
real spectrum Cross-real spectrum Cross-Correlated with modelCorrelated with modelspectrumspectrum
250250 500500 750750 10001000 12501250
2020
4040
6060
8080
100100x8x8
185.3185.3
255.7255.7
360.9360.9403.0403.0 519.1519.1
662.3662.3
805.5805.5
1007.41007.4
1155.51155.5
1226.81226.8
1324.81324.8
892.6892.6
m/zm/z
• Dynamic RangeDynamic Range
-Complex Peptide Complex Peptide MixturesMixtures
-Low StoichiometryLow Stoichiometry
• Tandem mass spectra Tandem mass spectra showing clearly the showing clearly the modified sitemodified site
Ab
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m/z
Phosphopeptide
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Identifying PTMs – LimitationsIdentifying PTMs – Limitations
Molecular Analysis of KinetochoreComposition and Organization
Cheeseman, Drubin, Barnes- UCBCheeseman, Drubin, Barnes- UCB
Additional Central Kinetochore Proteins
Ctf19p
Mcm21p
Okp1p
Ortiz et al. 1999
Mcm19p
Chl4p
Ghosh et al. 2001
** Defined by 2-hybrid and co-immunoprecipitation **
Ctf3p
Mcm22p
Mcm16p
Measday et al. 2002
The Ctf19p Complex
• 12 Subunits
NEW!
Mapped Phosphorylation Sitesin vivo
Phosphorylation Sites
13
1
4
0
2
3
Ipl1p Targets
6
1
3
0
1?
1?
Dam1p Complex -
Ndc80p Complex -
Ctf19p Complex -
Ipl1p Complex -
Bim1p Complex -
Mif2p Complex -
ComplexesComplexes
Direct Id of Protein Modifications in a Tandem Affinity Purified (TAP) Complex
C-terminally tagged atthe endogenous locus
ProtAcdc2
CBP
IgGSepharose
ProtA
cdc2CBP
Bind toIgG Beads
CalmodulinResin
cdc2CBP
Bind toCalmodulinResin
Elute w/ TEVCleavage
Elute w/ EGTA
Relative Quantitation of ProteinsRelative Quantitation of Proteins
• Metabolic labeling Metabolic labeling 1515NN– Chait et al. Chait et al. PNASPNAS 1999 1999
• Covalent labeling to introduce mass labelsCovalent labeling to introduce mass labels– CDCD33OH, CDOH, CD33CO-, CDCO-, CD22=CDCONH=CDCONH2 2
– James, Nicotinic-NHS DJames, Nicotinic-NHS D00/D/D44: : Anal. Chem.Anal. Chem. ‘00 ‘00
• 1818O labeling during Proteolytic Digestion O labeling during Proteolytic Digestion – Fenselau, Fenselau, Anal. ChemAnal. Chem 2001 2001
• Covalent labeling with affinity enrichmentCovalent labeling with affinity enrichment– Gygi et. al. Gygi et. al. Nature BiotechNature Biotech. 1999. 1999– Regnier Regnier J. ChromatographyJ. Chromatography 1999 1999
Large Scale Mass Spectrometry Analysis of Complexes
• Serial technique- throughput increases derive from adding additional instruments
• Analyses requiring high sensitivity and large dynamic range require more deliberate techniques- generally more manual
• LC/MS/MS 0.5-1hr• LC/LC/MS/MS 3hr-6 hr• Protein identification robust and accurate esp in
metazoans• Automation allows throughput increases, but
decreases sensitivity, dynamic range
Technology Advances for Mass Spectrometry of Complexes
• Throughput– Biochemical – Mass Spectrometry
• Sensitivity– Low copy number complexes– Low stoichiometry modifications– Identify proteins with fast on-off rates
• Direct Analysis of Intact Proteins– Isoforms– Modified forms
• Direct Analysis of Intact complexes– Stoichiometry– Shape
• H/D exchange to determine interaction surfaces
Comprehensive Analysis of Complex Protein Structures in the Cell
Total ProteinCharacterization
• Protein Identification: What’s there• Post Translational Modifications: Regulation• Quantification: Dynamics
Multiprotein Complex/OrganelleMultiprotein Complex/Organelle
Funding
• NIH NCRR RR11823 Yeast Resource Center, University of Washington
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